Compositions and methods for treatment of cancer using bacteria

ABSTRACT

Provided herein are compositions comprising substantially non-viable Gram-negative bacterial organisms that have a substantial reduction in endotoxin activity and/or pyrogenicity and methods for treating a cancer using the same. Also provided are methods for treating cancer provided herein, comprising administering to a mammal diagnosed with cancer, substantially non-viable Gram-negative bacteria having a substantial reduction in endotoxin activity and/or pyrogenicity, in an amount sufficient to inhibit growth or metastasis of the cancer. An additional method is provided comprising administering viable or non-viable Gram-negative bacterial organisms that have a genetic defect that results in a substantial loss of lipopolysaccharide within the outer membrane of the bacteria. Further provided are methods for reducing endotoxin activity and/or pyrogenicity in Gram-negative bacteria comprising treatment with polymyxin and glutaraldehyde.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.provisional application 61/748,369 filed on Jan. 2, 2013, which ishereby incorporated by reference.

FIELD

This disclosure relates to compositions comprising Gram-negativebacteria and methods for treating cancer by administering the same.

BACKGROUND

The association of cancer regression in patients undergoing bacterialinfection was observed and reported at least as early as 1868. Thesystemic administration of live attenuated Salmonella organisms to solidtumor bearing animals was reported to result in tumor therapy. See,e.g., U.S. Pat. No. 6,685,935 and Pawelek et al., (Lancet Oncol.4(9):548-56, 2003). Also, intravesical (non-systemic) administration ofattenuated Gram-positive mycobacteria (BCG) is approved in the UnitedStates for the treatment and prophylaxis of carcinoma in situ (CIS) ofthe urinary bladder.

Improvements in tumor therapy using live Gram-negative Salmonella havealso been reported for certain auxotrophic mutants. See e.g., Hoffman etal., (Amino Acids 37:509-521, 2009, U.S. Patent publication 20090300779(Zhao et al.), and Zhao et al. (Proc. Natl. Acad. Sci. (USA)102(3):775-760, 2005).

Salmonella having deletions in the msbB locus have been prepared whichexpress LPS lacking terminal myristoylation of lipid A in the outermembrane. TNF-alpha induction in mice and swine treated with thesemsbB-Salmonella strains was 33% and 14% of the amount induced bywild-type bacteria, respectively. See e.g., Low et al., Nature 17:37-41,1999 and U.S. Pat. No. 7,354,592 (Bermudes et al.). Administration ofsuch live organisms, including strain VNP20009, has been reported toinhibit the growth of subcutaneously implanted B16F10 murine melanoma,and the human tumor xenografts Lox, DLD-1, A549, WiDr, HTB177, andMDA-MB-231 grown in mice (Luo et al., Oncol. Res. 12(11-12):501-508,2001). Salmonella strain VNP20009 has also been reported to improve theanti-tumor efficacy of the chemotherapeutic agent cyclophosphamide atboth a maximum tolerated dose and with a low-dose metronomic regimen(Jia et al., Int. J. Cancer 121(3):666-674, 2007).

Conditional mutants of Gram-negative bacteria that cannot produce LipidA and that lack LPS in the outer membrane have been prepared but havebeen reported to be toxic to the organism. For example, mutationalinhibition of synthesis of 3-deoxy-D-manno-octulosonate (Kdo) ormutational inhibition of incorporation of Kdo molecules into lipidIV_(A) prevents lipid A and LPS synthesis and localization of LPSprecursors to the outer membrane of Gram-negative bacteria. Lipid IV_(A)is an LPS precursor that lacks glycosylation. Activation of thesemutations leads to loss of bacterial viability (Rick et al., Proc. Natl.Acad. Sci. USA 69(12):3756-3760, 1972, Belunis et al. J. Biol. Chem.270(46):27646-27652, 1995, and Taylor et al. J. Biol. Chem.275(41):32141-32146, 2000).

It is also possible to inhibit Kdo incorporation into lipid IV_(A),synthesis of lipid A and localization to the outer membrane through theuse of exogenously added compounds. Goldman et al. (J. Bacteriol.170(5):2185-91, 1988) describe antibacterial agents that specificallyinhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferaseactivity, thereby blocking the incorporation of 2-keto3-deoxy-D-manno-octulosonate (Kdo) into lipid IV_(A) of Gram-negativeorganisms. As LPS synthesis ceased, molecules similar in structure tolipid IV_(A) were found to accumulate, and bacterial growth ceased. Theauthors concluded that addition of Kdo to LPS precursor lipid speciesIV_(A) is the major pathway of lipid A-Kdo₂ formation in both S.typhimurium LT2 and Escherichia coli (E. coli).

More recently, mutants of Gram-negative bacteria have been prepared thatlack LPS, including lipid A or 6-acyl lipidpolysaccharide, in the outermembrane but maintain viability. For example, U.S. Patent publication2010/0272758 reports an E. coli K-12 strain KPM22 that is defective insynthesis of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). KPM22 has anouter membrane (OM) composed predominantly of lipid IV_(A). Viability ofthese organisms was achieved by the presence of a second-site suppressorthat facilitates transport of lipid IV_(A) from the inner membrane tothe outer membrane. This suppressor is reported to relieve toxicside-effects of lipid IV_(A) accumulation in the inner membrane andprovide sufficient amounts of LPS precursors to support OM biogenesis.The LPS precursor produced by this strain lacks endotoxin activity, asdetermined by its inability to induce TNF-alpha secretion by humanmononuclear cells at LPS precursor doses of up to 1 μg/mL. See also,Mamat et al., (Mol Microbiol. 67(3):633-48, 2008).

Dose-limiting side effects associated with infection and septic shocksignificantly limit systemic administration of live bacteria to cancerpatients. This limitation has been associated with wildtype bacteria(see e.g., Wiemann and Starnes, Pharmac. Ther. 64:529-564, 1994 forreview), and has also been associated with genetically attenuatedbacteria, which proliferate selectively in tumor tissue and expressmodified lipid A (see e.g., Toso et al., J. Clin. Oncol. 20(1):142-152,2002). These limitations have led to the use of heat killed bacteria forcancer therapy. See e.g., Havas et al. (Med. Oncol. & TumourPharmacother. 10(4):145-158, 1993), Ryoma et al. (Anticancer Res.24:3295-3302, 2004), Maletzki et al. (Clin. Develop. Immunol. 2012:1-16,2012), U.S. Pat. No. 8,034,359 B2 (Gunn), European Patent No. EP1,765,391 B1 (Gunn), and for review, Wiemann and Starnes (Pharmac. Ther.64:529-564, 1994). However, non-infectious, killed bacteria still inducesignificant dose-limiting toxicities associated with LPS-derivedendotoxin and other cell constituents, which are pyrogenic and canproduce symptoms of septic shock. Thus, further improvements in treatingcancer with bacteria are needed.

SUMMARY

Provided herein are compositions and methods for treating cancer in amammal (e.g., a human), diagnosed as having cancer, by administering tothat mammal an amount of Gram-negative bacteria wherein the bacteria are(i) non-viable or substantially non-viable in the mammal, (ii) have asubstantial reduction in endotoxin activity and/or pyrogenicity, and(iii) are administered in an amount sufficient to inhibit the growth ormetastatic potential of the cancer. In some embodiments, theGram-negative bacteria are rendered non-viable or substantiallynon-viable prior to administration to the mammal by treatment with (i)radiation, (ii) a chemical sterilant, (iii) an antibiotic thatinactivates endotoxin (e.g., polymyxin B or polymyxin E), or (iv) anantibiotic that disrupts the biosynthesis of KDO2-Lipid IV_(A).Alternatively, or in addition to, any one or more of the foregoingtreatments, the Gram-negative bacteria further comprises a geneticdefect that disrupts or partially disrupts the biosynthesis ofKDO2-Lipid IV_(A) or prevents the O-acylation of KDO2-Lipid IV_(A).Genetic defects that disrupt or partially disrupt the O-acylation ofKDO2-Lipid IV_(A) include, for example, defects which functionallydisrupt the msbB and lpxM loci.

In one aspect of the disclosure, compositions comprise substantiallynon-viable Gram-negative bacteria having a substantial reduction inendotoxin activity and/or pyrogenicity and a pharmaceutically acceptableexcipient. In one embodiment, the Gram-negative bacteria are madenon-viable by treatment with glutaraldehyde. In another embodiment, theendotoxin activity and/or pyrogenicity is reduced by treatment withpolymyxin B or polymyxin E. In a further embodiment, the endotoxinactivity and/or pyrogenicity is reduced by treatment withglutaraldehyde.

In another aspect, methods are provided to treat a mammal diagnosed ashaving cancer which included administering an amount of substantiallynon-viable Gram-negative bacteria having a substantial reduction inendotoxin activity and/or pyrogenicity, wherein the amount administeredis sufficient to inhibit growth or metastasis of the cancer.

In another aspect, the disclosure provides methods for treating cancerin a mammal (e.g., a human), diagnosed as having cancer, byadministering to that mammal an amount of Gram-negative bacteria whereinthe bacteria are viable, may or may not be attenuated, and have agenetic defect that results in a substantial or total loss oflipopolysaccharide within the outer membrane of the bacteria and whereinthe amount administered is sufficient to inhibit the growth ormetastatic potential of the cancer.

In one embodiment, the disclosure provides a method for treating acancer comprising administering to a mammal diagnosed as having canceran amount of viable or non-viable Gram-negative bacterial organisms thathave a genetic defect that results in a substantial loss oflipopolysaccharide within the outer membrane of the bacteria, whereinthe amount administered is sufficient to inhibit growth of the cancer.

In some embodiments, the genetic defect disrupts or partially disruptsthe biosynthesis of KDO2-Lipid IV_(A) or prevents the O-acylation ofKDO2-Lipid IV_(A).

In some embodiments, the cancer is a solid tumor.

In other embodiments, the mammal is further administered achemotherapeutic agent including, for example, cyclophosphamide. Inother embodiments, the mammal is further administered an antagonist ofan immune function-inhibiting receptor or receptor agonist including,for example, inhibiting the function of a T-cell receptor or T-cellreceptor ligand (e.g., CTLA-4, PD-1, PD-L1, and PD-L2).

In other embodiments, the mammal is further administered an agonist ofan immune function-stimulating receptor including, for example, agoniststhat stimulate a T-cell receptor. Suitable receptor targets include, forexample, GITR, 4-1BB, CD40, and OX40.

In other embodiments, the mammal is further administered an immunefunction-stimulating cytokine including, for example, interferon-alpha,interferon-beta, interferon-gamma, granulocyte-macrophagecolony-stimulating factor, interleukin-2, and interleukin-12.

In some embodiments, the Gram-negative bacteria are Salmonella orEscherichia.

In another embodiment, the disclosure provides for methods of killingand reducing endotoxin activity and/or pyrogenicity in Gram-negativebacteria by treating the bacteria with polymyxin B and glutaraldehyde.In one embodiment, viability is reduced to 0% and the endotoxin activityor pyrogenicity is reduced by about 90% or 96%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 demonstrate that incubation of E. coli with polymyxin B(PMB) reduces the level of bacterial cell-associated endotoxin activityand cell viability. This is further described in Example 2.

FIGS. 3 and 4 demonstrate that incubation of E. coli with glutaraldehyde(GA) reduces the level of bacterial cell-associated endotoxin activityand cell viability, as further described in Example 3.

FIG. 5 depicts transmission electron microscope images of E. coliuntreated (FIG. 5A), treated with 1,000 μg/mL PMB (FIG. 5B), 1% GA (FIG.5C), or both PMB and GA (FIG. 5D), demonstrating that the bacteriaremain intact after all treatments, as further described in Example 4.

FIG. 6 depicts a graph showing the dose-dependent effect ofPMB+GA-treated E. coli on the growth of subcutaneous murine B16F10melanoma in mice, as further described in Example 7.

FIG. 7 shows a graph showing the dose-dependent effect of untreated and1% GA-treated E. coli on the growth of subcutaneous murine B16F10melanoma in mice, as further described in Example 8.

FIGS. 8A and 8B illustrate graphs showing the dose-dependent effect ofPMB+GA-treated E. coli without and with metronomic cyclophosphamide(FIG. 8A) or anti-murine CTLA-4 antibody (FIG. 8B) on the growth ofsubcutaneous CT26 murine colorectal carcinoma in mice, as furtherdescribed in Example 9.

DETAILED DESCRIPTION

Provided herein are compositions comprising non-viable Gram-negativebacterial organisms and that have substantial reduction in endotoxinand/or pyrogenic activity and methods to treat cancer, comprisingadministering to a mammal suffering from cancer an amount of non-viableGram-negative bacterial organisms that have a substantial reduction inendotoxin or pyrogenic activity, wherein the amount administered issufficient to inhibit growth or metastasis of the cancer.

Possible mechanism(s) responsible for anti-tumor activity mediated bybacteria include selective proliferation of live bacterial organisms intumor tissue and stimulation of host immune responses, in particular viaLPS (endotoxin)-mediated induction of tumoricidal cytokine release fromhost mononuclear cells. However, the proliferation of live bacteria andLPS (endotoxin)-mediated induction of cytokines (even with LPSattenuated by msbB mutation), are believed responsible for dose-limitingtoxicity associated with treatment of mammals with live bacteria. Tosoet al. (J. Clin. Oncol. 20(1):142-152, 2002) treated cancer patientswith live msbB-attenuated Salmonella and dose-limiting toxicitiesincluded bacteremia and side-effects associated with cytokine release.Proliferation of bacteria in tumor tissue was lower and sensitivity tocytokine-mediated toxicities was higher than seen in human tumorxenograft models in mice. It is believed that systemic proliferation byviable bacteria and/or cytokine-related toxicities, mediated in part byLPS lacking one secondary acyl chain, may prevent administration of safeand effective doses of live, attenuated Gram-negative bacteria to somemammals (such as humans) other than mice, which are known to berelatively resistant to bacterial infection and associated septicconsequences of cytokine induction.

Although not wishing to be bound by theory, it is believed that killedor non-viable Gram-negative organisms with substantially reducedendotoxin activity and/or pyrogenicity can be administered to cancerpatients in amounts that are less toxic and more effective to treat thecancer as compared to using live or viable organisms, which proliferatein each patient's normal and tumor tissues in a variable manner thatcannot be controlled by the practitioner, either proliferatinginsufficiently to produce a therapeutic effect or proliferating toomuch, thereby producing unacceptable toxicity. It is also believed thatkilled or non-viable Gram-negative organisms with substantially reducedendotoxin activity and/or pyrogenicity can be administered to cancerpatients in amounts that are less toxic and more effective to treat thecancer as compared to using killed bacteria that express wildtype levelsof endotoxin activity and/or pyrogenicity.

It is also believed that viable Gram-negative organisms having a geneticdefect in the formation of LPS that results in a substantial reductionin the amount of glycosylated Lipid A and LPS in the outer membrane ofthe bacteria can be effective in the treatment of cancer whetheradministered alive and attenuated, so as to prevent furtherproliferation in the mammalian host, or as killed organisms. Althoughsuch organisms lack functional LPS molecules that cause endotoxic shockas well as provide a stimulus to the host's immune system, it isbelieved that there are other features of the Gram-negative bacteriathat will stimulate the host's innate or combined innate and adaptiveimmune responses to achieve tumor cell killing or tumor growthinhibition.

In one embodiment, the Gram-negative organisms used in cancer therapy,as disclosed herein, do not contain DNA that encodes or expressesnon-bacterial proteins (e.g., tumor-specific antigens). TheGram-negative organisms, therefore, are not a cancer vaccine in thatthey do not directly induce a specific immunological response against atumor antigen. Instead, these organism function as an adjuvant orbiological response modifier (BRM) that may generally stimulate the hostinnate immune response and possibly indirectly an adaptive anti-tumorimmune response. In some embodiments, the Gram-negative organisms areinjected directly in or near the site of the tumor, or are injectedsystemically and accumulate in or near the tumor. The increased innateimmune response against the organisms then may secondarily becomedirected against the tumor. In addition, or alternatively, immuneresponses against the organisms may stimulate or activate pre-existingtumor antigen-specific immune cells capable of participating in anadaptive anti-tumor response.

In an alternative embodiment, the Gram-negative organisms express DNAthat encodes for expression of non-bacterial proteins including, forexample, tumor-specific antigens or immune system stimulating proteins.Here again, the organisms may be injected in or near the tumor site, orsystemically, and induce an innate or adaptive immune response againstthe organism, the tumor-specific antigen, or both.

As used herein, the term tumor specific antigen refers to an antigenthat is expressed by a tumor but is not expressed by any normal cellsfrom the organism from which the tumor was derived. The termtumor-associated antigen refers to an antigen that is expressed by atumor but may also be expressed in a limited manner by normal cells fromthe organism from which the tumor was derived. The limited manner ofexpression may reflect a lower level of expression in normal cells thanthe tumor, expression by a limited type of normal cell or expression bynormal cells only during fetal development (i.e., a fetal antigen). Asused herein, an antigen is any molecule that can be recognized by animmune response, either an antibody or by an immune cell (e.g., T cell).

As used herein the terms “adjuvant” and “biological response modifier”refer to any substance that enhances an immune response to an antigen,tumor or tumor-associated cell. Thus, an adjuvant or biological responsemodifier is used to stimulate the immune system to respond morevigorously to a foreign antigen or a disease-causing ordisease-associated cell expressing a new antigen, or structurallyaltered or abnormal level of an existing antigen. However, in someembodiments, recombinant forms of Gram-negative bacteria that express,e.g., tumor specific or tumor-associated antigens or human immuneactivation proteins such as cytokines or chemokines are contemplated foruse in the disclosed methods. In an alternative embodiment, purifiedimmune activation proteins such as cytokines or chemokines are mixedwith the Gram-negative organisms prior to administration, or areadministered before or after the Gram-negative organisms.

As used herein the term mammal includes any mammal such as a human, dog,cat, cow, sheep, and the like. A preferred mammal is a human.

The term “Gram-negative bacteria” refers to bacteria that do not retainthe initial basic dye stain (e.g., crystal violet) that is part of theprocedure known as the Gram stain. In an exemplary Gram stain, cells arefirst fixed to a slide by heat and stained with a basic dye (e.g.,crystal violet), which is taken up by both Gram-negative andGram-positive bacteria. The slides are then treated with a mordant(e.g., Gram's iodine), which binds to basic dye (e.g. crystal violet)and traps it in the cell. The cells are then washed with acetone oralcohol, and then counterstained with a second dye of different color(e.g., safranin) Gram-positive organisms retain the initial violetstain, while Gram-negative organisms are decolorized by the wash solventorganic and hence show the counterstain. Exemplary Gram-negativebacteria include, but are not limited to, Escherichia spp., Shigellaspp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilusspp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp.,Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp.,Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp. andVibrio spp.

Within gram-negative organisms are the Enterobacteriaceae, a largefamily that includes, along with many harmless symbionts, manywell-known pathogens, such as Salmonella, E. coli, Yersinia pestis,Klebsiella and Shigella, Proteus, Enterobacter, Serratia, andCitrobacter. Members of the Enterobacteriaceae have been referred to asenterobacteria, as several members live in the intestines of animals.

Enterobacteriaceae are rod-shaped, typically 1-5 μm in length. They arefacultative anaerobes, fermenting sugars to produce lactic acid andvarious other end products. Most also reduce nitrate to nitrite andgenerally lack cytochrome C oxidase. Most have many flagella formotility, but some are nonmotile. Enterobacteriaceae arenonspore-forming.

The term “vector” refers to a nucleic acid molecule, which is capable oftransporting another nucleic acid to which it is linked as a singlepiece of nucleic acid. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors.” The term “expression system” as used herein refersto a combination of components that enable sequences in an expressionvector to be transcribed into RNA, folded into structural RNA, ortranslated into protein. The expression system may be an in vitroexpression system, such as is commercially available or readily madeaccording to known methods, or may be an in vivo expression system, suchas a eukaryotic or prokaryotic host cell that contains the expressionvector. In general, expression vectors useful in recombinant DNAtechniques can be “plasmids” which refer generally to circular doublestranded DNA that, in their vector form, is not bound to the bacterialchromosome. Other expression vectors well known in the art also can beused in expression systems (e.g., cosmid, phagemid and bacteriophagevectors).

The term “nucleic acid” refers to polynucleotides or oligonucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogsand as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

The term “modulation” as used herein refers to both upregulation (i.e.,activation or stimulation (e.g., by agonizing or potentiating)) anddownregulation (i.e., inhibition or suppression (e.g., by antagonizing,decreasing or inhibiting)). The term “inducible” refers in particular togene expression which is not constitutive but which takes place inresponse to a stimulus (e.g., temperature, heavy metals or other mediumadditive).

A. Candidate Bacterial Organisms

Candidate bacterial organisms that may be employed by the methods hereinare Gram-negative and are derived from those that have endotoxinactivity as wildtype organisms. Exemplary Gram-negative bacteriainclude, but are not limited to, Escherichia spp., Shigella spp.,Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp.,Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp.,Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp.,Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp. andVibrio spp. Candidate Gram negative organisms also may be those thatfall in the Enterobacteriaceae, Pseudomonadaceae, Neisseriaceae,Veillonellaceae, Bacteroidaceae, Vibrionaceae, Pasteurellaceae, andFusobacteriaceae families. In some embodiments, the candidate organismis a species of Salmonella or Escherichia spp.

One candidate Salmonella organism, VNP20009, has been described by Luoet al., Oncol Res. 12(11-12):501-8, 2001. VNP20009 is a geneticallymodified strain of Salmonella typhimurium with deletions in the msbB andpurI loci. Intravenous administration at doses ranging from 1×10⁴ to3×10⁶ cfu/mouse of live VNP20009 to tumor bearing mice inhibited thegrowth of subcutaneously implanted B16F10 murine melanoma, and the humantumor xenografts Lox, DLD-1, A549, WiDr, HTB177, and MDA-MB-231.VNP20009, given intravenously also inhibited the growth of lungmetastases in these animals. See also, U.S. Pat. No. 7,354,592 (Bermudeset al.).

Another candidate Salmonella organism is SL3235 described by Eisensteinet al. Med. Oncol. 12(2):103-8, 1995. SL3235 is an attenuated strain ofSalmonella that when administered live can cure plasmacytoma tumorgrowing in mice.

Further candidate Salmonella include auxotrophic mutants reported byHoffman et al., Amino Acids 37:509-521, 2009. The S. typhimurium A1-Rmutant is auxotrophic for leu-arg and has high anti-tumor virulence. Invitro, A1-R infects tumor cells and causes nuclear destruction. A1-Radministration treats metastatic human prostate and breast tumorsorthotopically implanted in nude mice. A1-R administered intravenously(i.v.) to nude mice with primary osteosarcoma and lung metastasis iseffective, especially against metastasis. A1-R also was reportedeffective against pancreatic cancer liver metastasis when administeredintrasplenically to nude mice. See also U.S. Patent publication20090300779 (Zhao et al.), and Zhao et al. (Proc. Natl. Acad. Sci. (USA)102(3):775-760, 2005).

A variety of Gram-negative organisms suitable for the treatment of solidtumors are reported in U.S. Pat. No. 6,685,935 (Pawelek et al.). Theseorganisms are referred to as super-infective as they replicatepreferentially in the tumor after administration. Included aresuper-infective, tumor-specific mutants of Salmonella spp., e.g.,Salmonella typhimurium. Also described are super-infective,tumor-specific mutants of Salmonella spp. containing a suicide gene suchas thymidine kinase from Herpes simplex virus, cytosine deaminase fromE. coli, or human microsomal p450 oxidoreductase. See also Pawelek etal., (Lancet Oncol. 4(9):548-56, 2003).

In one embodiment, E. coli is selected as the organism. One particularstrain contemplated is E. coli strain 2617-143-312, (Migula) Castellaniand Chalmers (ATCC® 13070™). Additional E. coli strains which may beused include MG1655 (ATCC® 47076) and KY8284 (ATCC® 21272).

The Gram-negative organisms used in the methods herein need not berecombinant organisms that contain or express DNA foreign to thewildtype form of the organism. However, in some embodiments, theorganisms may be modified to express some non-native molecules. Forexample, U.S. Pat. No. 7,452,531 reports preparation and use ofattenuated tumor-targeted bacteria vectors for the delivery of one ormore primary effector molecule(s) to the site of a solid tumor.According to the method, effector molecules, which may be toxic whenadministered systemically to a host, can be delivered locally to tumorsby attenuated tumor-targeted bacteria with reduced toxicity to the host.Specifically, the attenuated tumor-targeted bacteria can be afacultative aerobe or facultative anaerobe which is modified to encodeone or more primary effector molecule(s). The primary effectormolecule(s) include members of the TNF cytokine family, anti-angiogenicfactors, and cytotoxic polypeptides or peptides. The primary effectormolecules of the disclosure are useful, for example, to treat a solidtumor cancer such as a carcinoma, melanoma, lymphoma, sarcoma, ormetastases derived from these tumors.

B. Reducing Bacterial Endotoxin Activity

Various methods may be used to reduce endotoxin activity and/orpyrogenicity of bacterial organisms. As used herein, the term “endotoxinactivity” refers to portions of Gram-negative bacteria that can causetoxicity, including pyrogenicity and septic shock. The toxic effectsattributed to endotoxin have been found to be associated with theglycosylated lipid A portion of a lipopolysaccharide molecule present inor derived from the outer membrane of Gram-negative bacteria.

The term “Lipopolysaccharide” (LPS) refers to large molecules consistingof a lipid and a polysaccharide (glycophospholipid) joined by a covalentbond. LPS comprises three parts: 1) O antigen; 2) Core oligosaccharide,and 3) Lipid A. The O-antigen is a repetitive glycan polymer attached tothe core oligosaccharide, and comprises the outermost domain of the LPSmolecule. Core oligosaccharide attaches directly to lipid A and commonlycontains sugars such as heptose and 3-deoxy-D-mannooctulosonic acid(also known as KDO, keto-deoxyoctulosonate). Lipid A is a phosphorylatedglucosamine disaccharide linked to multiple fatty acids. The fatty acidsanchor the LPS into the bacterial membrane, and the rest of the LPSprojects from the cell surface. Bacterial death may result if LPS ismutated or removed.

Endotoxin activity resides in the lipid A domain portion of LPS. Whenbacterial cells are lysed by the immune system, fragments of membranecontaining lipid A are released into the circulation, causing fever(pyrogenicity), diarrhea, and a potentially fatal shock (calledendotoxic or septic shock). Toxicity of LPS is expressed by lipid Athrough the interaction with B-cells and macrophages of the mammalianimmune system, a process leading to the secretion of proinflammatorycytokines, mainly tumor necrosis factor (TNF), which may have fatalconsequences for the host. Lipid A also activates human T-lymphocytes(Th-1) “in vitro” as well as murine CD4+ and CD8+ T-cells “in vivo”, aproperty which allows the host's immune system to mount a specific,anamnestic IgG antibody response to the variable-size carbohydrate chainof LPS. On these bases, LPS has been recently recognized as a T-celldependent antigen “in vivo”.

Endotoxin activity can be measured by methods well known in the art,including, for example, the Limulus Amebocyte Lysate (LAL) assay, whichutilizes blood from the horseshoe crab, can detect very low levels ofLPS. The presence of endotoxin activity will result in coagulation ofthe limulus blood lysate due to amplification via an enzymatic cascade.Gel clotting, turbidometric, and chromogenic forms of the LAL assay arecommercially available. See, e.g., Lonza, Allendale, N.J., and ClongenLabs, Germantown, Md.

Enzyme linked immunoadsorbent assay (ELISA)-based endotoxin activityassays are also known such as the EndoLISA® from Hyglos, Munich area ofGermany. This assay employs an LPS specific phage protein attached tothe solid phase to capture LPS, and following a wash step, the presenceof LPS is determined by addition of recombinant Factor C, which whenactivated by LPS, cleaves a compound that then emits fluorescence.Factor C, present in the Limulus amebocyte lysate, normally exists as azymogen, and is the primer of the coagulation cascade that occurs in theLAL test.

Endotoxin activity can also be measured by evaluating induction ofTNF-alpha secretion, either from primary peripheral blood mononuclearcells in vitro, or by treating an animal with the suspected source ofendotoxin and measuring TNF-alpha levels in plasma, obtained from theanimal after approximately 1 to 4 hours. Primary mammalian peripheralblood mononuclear cells can be purchased from companies such as Lonza(Allendale, N.J., USA). TNF-alpha levels in cell supernatant or plasmacan be determined with ELISA kits, such as those available from ThermoScientific (Rockford, Ill., USA), Abcami (Cambridge, Mass., USA) oreBioscience (San Diego, Calif., USA).

Endotoxin activity can also be assessed in vivo by measuringpyrogenicity (rectal temperature increase) in rabbits in response tointravenously administered organisms or derivatives thereof.

The endotoxin activity and/or pyrogenicity of Gram-negative organismsmay be substantially reduced as compared to that of the wildtypeorganism. A substantial reduction in endotoxin activity is preferablymore than about 70%, more than about 75%, more than about 80%, more thanabout 85%, more than about 90%, more than 95% and more than about 99%.

Various methods are available to reduce the endotoxin activity ofGram-negative organisms. The methods include treatment of the organismswith an agent that binds to LPS or disrupts its formation, or bygenetically manipulating the bacterial organism to modify LPS or inhibitLPS formation.

In one embodiment, reduction in endotoxin activity or pyrogenicity isachieved by treating the bacterial organisms with an antibiotic thatinactivates endotoxin. A suitable such antibiotic is polymyxin B orpolymyxin E. For example, Cooperstock et al., Infect Immun. 1981 July;33(1):315-8, report that Polymyxin B treatment can reduce theinflammatory reactivity of LPS in vaccines of Gram-negative bacteriaincluding Bordetella pertussis, E. coli, Haemophilus influenzae, andPseudomonas aeruginosa. It is within the skill of one in the art todetermine the amount of antibiotic and conditions for treatment. In oneembodiment, the polymyxin, either polymyxin B or E, may be employed at aconcentration of approximately 3 micrograms to 5,000 micrograms per1×10⁷ to 5×10¹⁰ bacteria per milliliter. In another embodiment, theconcentration of polymyxin may be from about 200 micrograms to 5,000micrograms per 1×10⁷ to 5×10¹⁰ bacteria per milliliter. In oneembodiment, the antibiotic is applied to the bacteria for 10 minutes to4 hours or from about 30 minutes to about 3 hours. In one embodiment,the bacteria are grown in the presence of magnesium (Mg) in the form ofMgCl₂ and treated with polymyxin in the presence of MgCl₂, as well as ata temperature suitable to maintain the bacteria's integrity. In oneembodiment, the concentration of MgCl₂ in the growth medium is fromabout 0.5 mM to about 5.0 mM, or about 2 mM, and the concentration ofMgCl₂ in the treatment medium is from about 5.0 mM to about 30 mM, orabout 20 mM. In one embodiment, the temperature of the treatment mediumis from about 2° C. to about 10° C., or about 4° C. Bacterial integrityis determined by efficiency of recovery in a well-defined pellet aftercentrifugation at 3,000×g for 10 minutes, and by electron microscopy. Ina preferred embodiment, bacterial recovery after treatment and wash isgreater than about 80% and the bacteria appear intact by electronmicroscopy.

In another embodiment, reduction in endotoxin activity is achieved bytreating the bacterial organisms with an antibiotic known to disrupt thebiosynthesis of KDO2-Lipid IV_(A). For example, Goldman et al., J.Bacteriol. 170(5):2185-91, 1988 describe antibacterial agents, includingantibacterial agent III, which specifically inhibitCTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity andwhich are useful to block the incorporation of3-deoxy-D-manno-octulosonate (KDO) into LPS of Gram-negative organisms.As LPS synthesis ceased, bacterial growth ceased. The addition of KDO toLPS precursor species lipid IV_(A) is the major pathway of lipid A-KDOformation in both S. typhimurium and E. coli. In one embodiment, theantibiotic is antibacterial agent III and Gram-negative bacteria aretreated with a suitable amount, such as, for example 5 micrograms permilliliter to 500 micrograms per milliliter for a suitable time, forexample 2 to 8 hours.

A reduction in endotoxin activity may be achieved by introducing agenetic defect into the organism. The term “defect” as used herein, withregard to a gene or expression of a gene, means that the gene isdifferent from the normal (wildtype) gene or that the expression of thegene is at a reduced level of expression compared to that of thewildtype gene. The defective gene may result from a mutation in thatgene, or a mutation that regulates the expression of that gene. (e.g.,transcriptional or post-transcriptional)

In one embodiment, a reduction in endotoxin activity may be achieved byintroducing a genetic defect that disrupts the biosynthesis ofKDO2-Lipid IV_(A). For example, Woodard et al., U.S. Patent publication20100272758, report viable non-toxic Gram-negative bacteria (e.g., E.coli) substantially lacking LPS within the outer membrane. The authorsdescribe E. coli K-12 strain KPM22 as defective in synthesis of3-deoxy-d-manno-octulosonic acid (Kdo). KPM22 has an outer membrane (OM)composed predominantly of lipid IV_(A), an LPS precursor that lacksglycosylation. Viability of the organisms is achieved by the presence ofa second-site suppressor that transports lipid IV_(A) from the innermembrane (IM) to the outer membrane. This suppressor is reported torelieve toxic side-effects of lipid IV_(A) accumulation in the innermembrane and provide sufficient amounts of LPS precursors to support OMbiogenesis. See also, Mamat et al., (Mol Microbiol. 67(3):633-48, 2008).

In another embodiment, Bramhill et al., U.S. Patent Publication2011-0224097, describe viable Gram-negative bacteria comprising outermembranes that substantially lack a ligand, such as Lipid A or 6-acyllipopolysaccharide that acts as an agonist of TLR4/MD2.

According to Bramhill, the bacteria may comprise reduced activity ofarabinose-5-phosphate isomerases and one or more suppressor mutations,for example in a transporter thereby increasing the transporterscapacity to transport Lipid IVA, or in membrane protein YhjD. One ormore genes (e.g., IpxL, IpxM, pagP, IpxP, and/or eptA) may besubstantially deleted and/or one or more enzymes (e.g., LpxL, LpxM,PagP, LpxP, and/or EptA) may be substantially inactive.

In another embodiment, a reduction in endotoxin activity may be achievedby introducing a genetic defect that prevents synthesis of Kdo. Forexample, Rick et al., (Proc Natl Acad Sci USA. 69(12):3756-60, 1972)report an auxotrophic mutant of Salmonella typhimurium that is defectivein the synthesis of the 3-deoxy-D-mannooctulosonate (ketodeoxyoctonate)region of the LPS and requires D-arabinose-5-phosphate for growth. Themutant defect was due to an altered ketodeoxyoctonate-8-phosphatesynthetase (kdsA) with an apparent K(m) for D-arabinose-5-phosphate35-fold higher than that of the parental enzyme. This caused the mutantstrain to be dependent on exogenous D-arabinose-5-phosphate both forgrowth and for synthesis of a complete LPS. In another example, Beluniset al., (J. Biol. Chem. 270(46):27646-27652, 1995) disrupted the Kdotransferase (kdtA) gene in E. coli, which prevented incorporation of Kdointo lipid IV_(A). This mutation was lethal, but could be rescued by theconditional presence of a temperature-sensitive plasmid encoding kdtA.The development of conditional mutants in the Kdo synthesis pathwayallows for growth of the bacteria, followed by transfer to thenon-permissive condition, resulting in sufficient growth or survival toproduce non-viable bacteria with significantly reduced endotoxinactivity.

In addition to LPS-derived endotoxin, various other constituents ofGram-negative organisms can induce or contribute to pyrogenicity andseptic shock, including outer membrane proteins, fimbriae, pili,lipopeptides, and lipoproteins (reviewed by Jones, M., Int. J. Pharm.Compd., 5(4):259-263, 2001). Pyrogenicity can be measured by a rabbitmethod, well known in the art, involving assessment of rectaltemperature after intravenous administration of putative pyrogens.

It has been found that treatment of a Gram-negative organism with acombination of polymyxin B and glutaraldehyde produced a 30-foldreduction in pyrogenicity, as measured in rabbits. In one embodiment,1,000 micrograms per milliliter (μg/mL) of polymyxin B and 1%glutaraldehyde was employed to produce a 30-fold reduction inpyrogenicity, as measured in rabbits. The pyrogenicity is reduced by acombination of polymyxin B reaction with LPS and glutaraldehydereactivity with LPS and/or other bacterial constituents. Theglutaraldehyde serves a dual role in this setting by also killing thebacteria. Thus, in one embodiment is provided a method of reducingendotoxin activity and pyrogenicity of and killing a Gram-negativebacterial microorganism by treating said bacteria with a combination of1,000 μg/mL polymyxin B and 1% glutaraldehyde. In another embodiment,the Gram-negative bacteria are treated with a combination of polymyxin Bat a dose range between about 3 μg/mL to about 1,000 μg/mL andglutaraldehyde at a dose range between about 0.1% to about 1.0%. In afurther embodiment, the dose range of polymyxin B is between about 100μg/mL to about 1,000 μg/mL and glutaraldehyde is at a dose range betweenabout 0.5% to about 1.0%. Additionally, Gram-negative bacteria may betreated, for example with a dose range of polymyxin B between about1,000 μg/mL to about 3,000 μg/mL and glutaraldehyde is at a dose rangebetween about 0.5% to about 1.0%. In another aspect, Gram-negativebacteria maybe treated, for example with a dose range of polymyxin Bbetween about 3,000 μg/mL to about 5,000 μg/mL and glutaraldehyde is ata dose range between about 0.5% to about 2.0%. In one embodiment, theendotoxin activity is reduced by about 70%, or about 75%, or about 80%,or about 85%, or about 90%, or about 92%, and pyrogenicity is reduced byabout 75%, or about 80%, or about 85%, or about 90%, or about 95%, orabout 97%.

C. Rendering Bacteria Non-Viable

Bacteria for administration according to the methods of the disclosureare rendered non-viable or substantially non-viable either prior toadministration or become so upon administration. What is meant by“non-viable” is that the organisms are killed by treatment with anexogenous agent, and/or contain a mutation that results in an inabilityof the organisms to survive in a mammalian host. Substantiallynon-viable bacteria are strains that have had their viability reduced byat least 80%, 85%, 90%, 95%, 99%, or more. In preferred embodiments forbacteria that are not killed or not completely killed, the bacteria arefurther treated or modified such that they cannot proliferate within amammalian host. In some embodiments where LPS is substantially notproduced, it is contemplated that non-viable, attenuated, or viablebacteria are administered.

Preferred methods of rendering bacteria non-viable are treatment with acompound that binds to LPS, thereby blocking its endotoxin activity, ortreatment with a compound that interferes with LPS biosynthesis. In bothcases, LPS binding and interference with LPS synthesis, viability isreduced as a result of permeabilization of the cell envelope. Anotherapproach is to grow bacterial strains with conditional mutations in theLPS biosynthesis pathway that are suppressed during growth and thentransfer to a non-permissive condition which activates the mutation anddisrupts LPS biosynthesis. In each instance, the procedure applied isone that renders the bacteria non-viable by, determining in eachsetting, the optimal time of treatment or dose of compound, such thatviability has been substantially lost with retention of significantbacterial cell integrity. In the case where non-viability is less than100%, bacteria can be used which contain a mutation preventing furtherproliferation of viable bacteria in a mammalian host (e.g. adiaminopimelic acid auxotroph, as described by Bukhari and Taylor, J.Bacteriol. 105(3):844-854, 1971 and Curtiss et al., Immunol. Invest.18(1-4):583-596, 1989).

If alternative or additional methods of rendering bacteria non-viableare desired, a preferred method for killing bacteria is ionizingradiation (gamma rays or electron beam), but could also be done by otherstandard sterilization methods such as moist or dry heat, sterilant gasor vapor (see, e.g., Shintani et al., Biocontrol Science, 16(3):85-94,2011). Additional non-standard methods of terminal sterilization thatcould be used include chemical treatment such as a chemical sterilant,and are summarized by Rutala and Weber (Emerg. Infect. Dis.7(2):348-353, 2001) and Yaman (Curr. Opin. Drug Discov. Develop.4(6):760-763, 2001). Examples of chemical gas, vapor and liquidsterilants include ethylene oxide gas (EOG), chlorine dioxide, vaporousphase of liquid hydrogen peroxide (VHP), formaldehyde, glutaraldehyde(e.g., ≧0.05% for ≧10 minutes), ortho-phthalaldehyde (OPA) (e.g. ≧0.1%for ≧5 minutes), and phenol. Methods that kill bacteria may affect theintegrity of the organism. For example, the addition of heat may damagebacterial integrity, as opposed to the use of radiation. Reference to abacterial organism as used herein includes the fully intact organism andpartially degraded forms of the organism that may arise when theorganisms are killed, but does not extend to subcellular fractions ofthe organisms that have become separated from other cellular components,such as a cell wall fraction (preparation) or a cell wall skeleton (seee.g., U.S. Pat. No. 4,436,727), cytoplasmic fraction, and the like.

D. Compositions

In one embodiment, is provided a composition comprising non-viableGram-negative bacterial organisms having a substantial reduction inendotoxin and/or pyrogenic activity and a pharmaceutically acceptableexcipient. In another embodiment, at least about 80% of the organismsare non-viable or at least about 90% of the organisms are non-viable, orabout 100% of the organisms are non-viable. In one embodiment, theorganisms have their viability reduced by about 80%, or by about 85%, orby about 90%, or by about 95%, or by about 100%.

In one embodiment, the endotoxin and/or pyrogenic activity is reduced byabout 70%, or by about 75%, or by about 80%, or by about 85%, or byabout 90%, or by about 95%. The composition may contain any contemplatedamount of non-viable or viability-reduced organisms in combination withany contemplated reduction in endotoxin or pyrogenic toxicity. Inanother embodiment, the composition comprises at least about 100%non-viable organisms having at least about 95% reduced endotoxinactivity and pyrogenicity.

Compositions described herein may be formulated in a variety of ways foruse in the methods described herein. In one embodiment, the compositioncomprises the organisms as described throughout and a pharmaceuticallyacceptable carrier.

“Pharmaceutically acceptable carriers” refers to any diluents,excipients, or carriers that may be used in the compositions.Pharmaceutically acceptable carriers include ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances, such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They are selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

The pharmaceutical compositions may be manufactured by methods wellknown in the art such as microbial growth in fermenters, followed byconcentration and washing by centrifugation, filtration or dialysis,conventional granulating, mixing, dissolving, encapsulating,lyophilizing, or emulsifying processes, among others. Compositions maybe produced in various forms, including granules, precipitates, orparticulates, powders, including freeze dried, rotary dried or spraydried powders, amorphous powders, injections, emulsions, elixirs,suspensions or solutions. Formulations may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these.

Pharmaceutical compositions may be prepared as liquid suspensions orsolutions using a sterile liquid, such as oil, water, alcohol, andcombinations thereof. Pharmaceutically suitable surfactants, suspendingagents or emulsifying agents, may be added for oral or parenteraladministration. Suspensions may include oils, such as peanut oil, sesameoil, cottonseed oil, corn oil and olive oil. Suspension preparation mayalso contain esters of fatty acids, such as ethyl oleate, isopropylmyristate, fatty acid glycerides and acetylated fatty acid glycerides.Suspension formulations may include alcohols, such as ethanol, isopropylalcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, suchas poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil andpetrolatum, and water may also be used in suspension formulations.

The compositions are formulated for pharmaceutical administration to amammal, preferably a human being. Such pharmaceutical compositions ofthe invention may be administered in a variety of ways, includingparenterally. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

Sterile injectable forms of the compositions may be aqueous oroleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation. Compositions may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion.

E. Methods for Treating Cancer

Cancers suitable for treatment by the methods herein include generallycarcinomas, leukemias or lymphomas, and sarcomas. Carcinomas may be ofthe anus, biliary tract, bladder, breast, colon, rectum, lung,oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, kidney,gallbladder and bile ducts, small intestine, urinary tract, femalegenital tract, male genital tract, endocrine glands, thyroid, and skin.Other suitable cancers include carcinoid tumors, gastrointestinalstromal tumors, head and neck tumors, unknown primary tumors,hemangiomas, melanomas, malignant mesothelioma, multiple myeloma, andtumors of the brain, nerves, eyes, and meninges.

In some embodiments, the cancers to be treated form solid tumors, suchas carcinomas, sarcomas, melanomas and lymphomas.

Cancer therapy, as described herein is achieved by administering anamount of Gram-negative (live or dead as appropriate) organisms that issufficient to inhibit growth or metastasis of the cancer. As employedherein, the phrase “a sufficient amount,” refers to a dose (or series ofdoses) sufficient to impart a beneficial effect on the recipientthereof. The specific therapeutically effective dose level for anyparticular subject will depend upon a variety of factors including thetype of cancer being treated, the severity of the cancer, the activityof the specific organism or combined composition, the route ofadministration, the rate of clearance of the organism or combinedcomposition, the duration of treatment, the drugs (if any) used incombination with the organism, the age, body weight, sex, diet, andgeneral health of the subject, and like factors well known in themedical arts and sciences. Various general considerations taken intoaccount in determining the “therapeutically effective amount” are knownto those of skill in the art and are described, e.g., in Gilman et al.,eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics,8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Co., Easton, Pa., 1990. Dosage levelstypically fall in the range of about 0.001 up to 100 mg/kg/day; withlevels in the range of about 0.05 up to 10 mg/kg/day being generallyapplicable for compounds. Dosage levels for administered organismstypically fall in the range of about 10⁶ to 10¹² per m². A compositioncan be administered parenterally, such as intravascularly,intravenously, intraarterially, intramuscularly, subcutaneously, orallyor the like. Bacterial organisms can be administered parenterally, suchas intravascularly, intravenously, intraarterially, intramuscularly,subcutaneously, intraperitoneally, or intravesically.

A therapeutically effective dose can be estimated by methods well knownin the art. Cancer animal models such as immune-competent mice withmurine tumors or immune-compromised mice (e.g. nude mice) with humantumor xenografts are well known in the art and extensively described inmany references incorporated for reference herein. Such information isused in combination with safety studies in rats, dogs and/or non-humanprimates in order to determine safe and potentially useful initial dosesin humans. Additional information for estimating dose of the organismscan come from studies in actual human cancer. For example, Toso et al.(J Clin Oncol. 20(1):142-52, 2002) report a phase I clinical trial inwhich live VNP20009 was administered to patients with metastaticmelanoma. Patients received 30-minute intravenous bolus infusionscontaining 10(6) to 10(9) cfu/m(2) of VNP20009. The maximum-tolerateddose was 3×10(8) cfu/m(2). Dose-limiting toxicity was observed inpatients receiving 1×10(9) cfu/m(2), which included thrombocytopenia,anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting,nausea, elevated alkaline phosphatase, and hypophosphatemia.

The organisms may be administered as a pharmaceutically acceptableformulation. The term “pharmaceutically acceptable” means a materialthat is not biologically or otherwise undesirable, i.e., the materialmay be administered to an individual along with the selected organism orcombined compound without causing any undesirable biological effects orinteracting in a deleterious manner with any of other administeredagents. This is more thoroughly described above.

The term “treating” a subject for a condition or disease, as usedherein, is intended to encompass curing, as well as ameliorating atleast one symptom of the condition or disease. Cancer patients aretreated if the patient is cured of the cancer, the cancer goes intoremission, survival is lengthened in a statistically significantfashion, time to tumor progression is increased in a statisticallysignificant fashion, there is a reduction in lymphocytic orhematopoietic tumor burden based on standard criteria established foreach type of lymphocytic or hematopoietic malignancy, or solid tumorburden has been decreased as defined by response evaluation criteria insolid tumors (RECIST 1.0 or RECIST 1.1, Therasse et al. J. Natl. CancerInst. 92(3):205-216, 2000 and Eisenhauer et al. Eur. J. Cancer45:228-247, 2009). As used herein, “remission” refers to absence ofgrowing cancer cells in the patient previously having evidence ofcancer. Thus, a cancer patient in remission is either cured of theircancer or the cancer is present but not readily detectable. Thus, cancermay be in remission when the tumor fails to enlarge or to metastesize.Complete remission as used herein is the absence of disease as indicatedby diagnostic methods, such as imaging, such as x-ray, MRI, CT and PET,or blood or bone marrow biopsy. When a cancer patient goes intoremission, this may be followed by relapse, where the cancer reappears.

The term “substantially” unless indicated otherwise means greater thanabout 80%, greater than about 90%, greater than about 95% and greaterthan about 99%.

F. Combinations for Treating Cancer

The methods of cancer therapy described herein may employ administrationof Gram-negative organisms together with one or more antagonists ofreceptors or ligands that negatively modulate the host immune response.Antagonists may be directed to PD-1, PD-L1 or CTLA-4 and typically areadministered intravenously, for example at a dose range of about 0.03milligram per kilogram to about 30 milligram per kilogram every 1 to 4weeks.

Programmed cell death protein 1 (PD-1) is a protein that in humans isencoded by the PDCD1 gene. PD-1 has also been designated as CD279(cluster of differentiation 279). PD-1 is a type I membrane protein of268 amino acids. PD-1 is a member of the extended CD28/CTLA-4 family ofT cell regulators. See, e.g., Ishida et al., EMBO J. 11 (11): 3887-95,1992. The proteins contain an extracellular IgV domain followed by atransmembrane region and an intracellular tail. The intracellular tailcontains two phosphorylation sites within in an immunoreceptortyrosine-based inhibitory motif and an immunoreceptor tyrosine-basedswitch motif. This suggests that PD-1 negatively regulates TCRsignaling. PD-1 is expressed on the surface of activated T cells, Bcells, and macrophages. PD-1 is a broad negative regulator of immuneresponses.

PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7family. See, e.g., Freeman et al., J. Exp. Med. 192 (7):1027-34, 2000and Latchman et al., Nat. Immunol. 2(3): 261-8, 2001. PD-L1 is a 40 kDatype 1 transmembrane protein that has been reported to play a major rolein suppressing the immune system during pregnancy, tissue allografts,autoimmune disease and hepatitis. PD-L1 protein is upregulated onmacrophages and dendritic cells (DC) in response to LPS and GM-CSFtreatment, and on T cells and B cells upon TCR and B cell receptorsignaling. The formation of a PD-1 receptor/PD-L1 ligand complextransmits an inhibitory signal which reduces the proliferation of CD8+ Tcells (during an immune response) at the lymph nodes and PD-1 also cancontrol the accumulation of foreign antigen specific T cells in thelymph nodes through apoptosis. PD-L2 expression is more restricted andis expressed mainly by dendritic cells and a few tumor lines.

CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), also known as CD152 (Clusterof differentiation 152), is a protein receptor that downregulates theimmune system. CTLA-4 is expressed on the surface of helper, effectorand immunoregulatory T-cells, which lead the cellular immune attack onantigens. The T cell can be turned on by stimulating the CD28 receptoror turned off by stimulating the CTLA-4 receptor. CTLA-4, like that ofthe T-cell co-stimulatory protein, CD28, bind to CD80 and CD86, alsocalled B7-1 and B7-2, respectively, on antigen-presenting cells. T-cellactivation through the T-cell receptor and CD28 leads to increasedexpression of CTLA-4, an inhibitory receptor for B7 molecules.

Enhancing or prolonging T-cell activation has been achieved bymonoclonal antibodies (mAbs) to CTLA-4 and PD-1. Ipilimumab andtremelimumab are monoclonal antibodies that inhibit CTLA-4, and havebeen shown to induce or enhance anti-tumor immune responses leading todurable anti-tumor effects. Ipilimumab (also known as MDX-010 orMDX-101), marketed in the U.S. under the name Yervoy, is sold by BristolMyers Squibb for the treatment of unresectable or metastatic malignantmelanoma. BMS-936558 (MDX-1106) is a monoclonal antibody against PD-1and has exhibited significant anti-tumor activity in human clinicaltrials. See, e.g., Brahmer et al., J. Clin. Oncol., 28(19):3167-3175,2010, Brahmer et al., N. Engl. J. Med., 366(26):2455-2465, 2012; andLipson et al., Clin. Can. Res. 19(2):462-468, 2013.

Inhibition of CTLA-4 also may be achieved by a fusion protein (CTLA4Ig)made up of CTLA-4 and Fc of immunoglobulin (Ig) heavy chain. See, e.g.,Park et al., Pharm Res. 20(8):1239-48, 2003.

An additional important negative regulator of the immune response in thetumor microenvironment is the signal transducer and activator oftranscription (STAT) signal responsive transcription factor STAT3.Activity of this factor is elevated in tumor and associated immunecells. STAT3 activity in tumor cells contributes to enhanced survival,proliferation, invasion and metastasis, as well as stimulation ofangiogenesis. Elevated STAT3 activity in immune cells leads toaccumulation and activation of immunosuppressive cells, such as Treg,Th17 and myeloid derived suppressor cells within the tumormicroenvironment. See e.g., Rébé et al. (JAK-STAT 2(1):e23010-1-10,2013) for review. The widely used type 2 diabetes drugs metformin andphenformin have been shown to have antitumor activity and the mechanismis thought to include inhibition of STAT3 activity, resulting indecreased anti-tumor immunosuppression. See e.g., Deng et al., (CellCycle 11(2):367-376, 2012), Hirsch et al., (Proc. Natl. Acad. Sci., USA110(3):972-977, 2013), Appleyard et al., (British J Cancer106:1117-1122, 2012), Jiralerspong et al., (J Clin Oncol.27(20):3297-3302, 2009), and Del Barco et al., (Oncotarget2(12):896-917, 2011) for review. The methods of cancer therapy describedherein may employ administration of Gram-negative organisms togetherwith an inhibitor of STAT3 expression or activity. Such inhibitors mayinclude metformin and phenformin. Metformin maybe administered, forexample at a dose range of between about 50 milligrams to about 1,000milligrams, usually 1 to 3 times per day. Phenformin is typicallyadministered at a dose range of between about 20 milligrams to about 800milligrams 1 to 2 times per day.

The methods of cancer therapy described herein may also employadministration of Gram-negative organisms together with one or moreagonists of receptors or ligands that positively modulate the hostimmune response. Agonists directed to 4-1BB (CD137), GITR, CD40 or OX40(CD134) and can be administered, for example intravenously at a doserange of between about 0.03 milligram per kilogram to about 30 milligramper kilogram every 1 to 4 weeks.

Glucocorticoid inducible tumor necrosis factor receptor (TNFR)-relatedprotein (GITR), 4-1BB (CD137), CD40 and OX40 (CD134) are costimulatoryTNFR family members that are expressed on regulatory and effector Tcells as well as on other cells of the immune system. Activation ofthese proteins leads to stimulation or enhancement of immune function.Activating monoclonal antibodies for each of these proteins haveexhibited anti-tumor activity in preclinical models and have enteredclinical development. See, e.g., Melero et al., Clin. Cancer Res.15(5):1507-1509, 2009, Garber, JNCI-103(14):1079-1082, 2011, Khong etal., Int. Rev. Immunol. 31(4):246-266, 2012, Vinay and Kwon, Mol.Cancer. Ther. 11(5):1062-1070, 2012, Snell et al., Immunol. Rev.244(1):197-217, 2011, and So et al., Cytokine Growth Factor Rev.19(3-4):253-262, 2008.

The methods of cancer therapy described herein may also employadministration of Gram-negative organisms together with one or morechemotherapeutic agents. Such agents may include cyclophosphamide. It iscontemplated that when cyclophosphamide is used in the methods describedherein, it may administered in a dose of between 5 mg/m² to 750 mg/m²intravenously or orally daily or every 21 days. Alternatively,cyclophosphamide may be administered, for example, in a metronomicregimen at a dose of between 5 mg to 100 mg orally daily. See, forexample, Jia et al., Int. J. Cancer 121(3):666-674, 2007.

Stimulation of anti-tumor immune responses has been demonstrated withvarious cytokines. See, for example, Smyth et al., Immunological Rev.202:275-293, 2004 and Kim-Schulze, Surg. Oncol. Clin N. Am. 16:793-818,2007 for reviews. The methods of cancer therapy described herein mayalso employ administration of Gram-negative organisms together withrecombinantly expressed or isolated and purified cytokines, such asinterferon-alpha, interferon-beta, interferon-gamma,granulocyte-macrophage colony-stimulating factor, interleukin-2, andinterleukin-12.

The methods of cancer therapy described herein may also employGram-negative bacteria administered together with recombinantlyexpressed or isolated and purified interferon-alpha. Theinterferon-alpha may be administered either subcutaneously,intramuscularly, or intravenously at a dose range of between about 3×10⁵to about 3×10⁸ IU 1, 3, 5 or 7 times per week. In another embodiment,Gram-negative bacteria may be administered together withinterferon-beta. In certain embodiments, the interferon-beta will beadministered subcutaneously or intravenously at a dose range of betweenabout 0.01 milligrams to about 5 milligrams either once a week or everyother day. Interferon-gamma may also be co-administered. In oneembodiment, the interferon-gamma may be administered eithersubcutaneously or intravenously at a dose range of between about 1×10⁵IU to about 1×10⁹ IU either once or daily.

In additional methods, interleukins (e.g. interleukin-2, andinterleukin-12) may be co-administered. In one embodiment, interleukinsmay be administered intravenously in a dose of between about 1×10⁴ toabout 1×10⁷ IU once per week or up to three times a day in combinationwith Gram-negative bacteria. Additional methods include Gram-negativebacteria being administered, for example with Granulocyte-macrophagecolony-stimulating factor either subcutaneously, intradermal, orintravenously typically at a dose range of between about 5 micrograms toabout 5 milligrams, either daily or monthly. In any of the combinationtreatments noted throughout, it is contemplated the organisms may beadministered before or after the additional cancer treatment. They mayalso be administered concurrently.

The following examples serve to illustrate the present disclosure. Theseexamples are in no way intended to limit the scope of the disclosure.

EXAMPLES Example 1

Optimal conditions for inactivation of lipopolysaccharide-associatedendotoxin activity and bacterial cell killing by polymyxin B withoutloss of cell integrity are determined for each bacterial strain byincubating concentrated late log bacteria (10⁹ to 10¹¹ per mL) at 37° C.in phosphate buffered saline (PBS) with 1-100 μg/mL of polymyxin B forvarious times between 2 minutes and 6 hours. Viability is determined byserial dilution plating of control and treated bacterial suspensions ongrowth-compatible agar plates, followed by overnight incubation andcolony counting. Cell integrity is determined by visual (microscope)examination and analysis of absorbance at 600 nm. Endotoxin activity isdetermined by the Limulus Amebocyte Lysate (LAL) assay. Soluble orexcess polymyxin and cell debris, including soluble endotoxin, areremoved by centrifugation-mediated washing with 0.9% NaCl (normalsaline).

Alternatively, optimal conditions for isolation of intact, non-viablebacteria with defective LPS, resulting from a conditional mutation, aredetermined as described for polymyxin treatment, except that bacteriaare grown in LB (Lysogeny broth) medium under the non-permissivecondition and removed at various times, followed by analysis andprocessing as described for polymyxin treatment.

Polymyxin-treated bacteria or saline-washed late log phase LPSmutant/defective bacteria are freeze-dried using trehalose as thecryoprotectant (see, e.g., Leslie et al., App. Environment. Microbiol.61(10):3592-3597, 1995; Gu et al., J. Biotech. 88:95-105, 2001 andAmerican Type Culture Collection Bacterial Culture Guide). If desired,bacterial viability is further reduced by treatment with ionizingradiation at a dose sufficient to reduce viability to 0%, without lossof bacterial integrity.

Freeze-dried bacteria are resuspended in sterile water prior to use inanti-tumor studies. PBS-washed murine tumor cells (B16 and B16F10melanoma, CT-26 colorectal carcinoma, Panc02 pancreatic carcinoma orLewis Lung carcinoma (10⁵-10⁷ cells, depending on cell line) areimplanted subcutaneously on the back of shaved C57BL/6 mice. Mice arerandomized and treatment is initiated when tumors can be first palpated,when tumors have reached an average volume of 75 mm³, or when tumorshave reached an average volume of 300 mm³ (as estimated by calipermeasurement). Resuspended bacteria are injected once to twice per weekvia the tail vein or intraperitoneally (i.p.) at individual dosesranging from 10³ to 10¹⁰ per 0.1-0.2 mL injection volume. Antibodyantagonists or agonists directed to T-cell receptors are administeredi.p. at individual doses of 3-100 micrograms once to twice per week.Cyclophosphamide is administered i.p. at up to 150 mg/kg every other dayfor 5 days (MTD dosing) or at 25 mg/kg per day in the drinking water(metronomic dosing). Mice are weighed twice per week and clinicalobservations are recorded. Tumor measurements (by caliper) are carriedout twice per week and mice are humanely sacrificed if/when tumors reach1,000 mm³, become necrotic or if ≧15% weight loss is observed. Tumorsare removed and weighed, and minimal necropsy is carried out withsacrificed mice. Mice may be re-challenged with tumor cell implantationif long-term tumor regression or cures are observed.

Example 2

In Example 2, E. coli strain 2617-143-312 (Migula) Castellani andChalmers (ATCC® 13070™) were used. This non-hazardous Gram-negativebacterium requires exogenous diaminopimelic acid (DAP) for growth. Sincemammals do not make DAP, this bacterial strain is not viable and cannotcause infections in mammals. In addition, the DAP auxotrophy can be usedto monitor contamination during in vitro studies. Bacteria were grown tolate log phase (based on O.D.₆₀₀) in LB Miller broth with 2 mM MgCl₂,0.5% glucose and 1 mM DAP at 37° C. with constant shaking at 300 rpm.The culture was washed three times by centrifugation at 2,000×g for 15minutes and resuspension in 4° C. LB Miller broth containing 20 mMMgCl₂, 0.5% glucose and 0.1 mM DAP (PMB treatment medium). Finalresuspension was made at 2×10¹⁰ bacteria per mL, based on an O.D.₆₀₀ of1 being equal to 1.12×10⁹ bacteria per mL. Individual aliquots of theculture were incubated without and with various concentrations ofPolymyxin B (PMB) (Calbiochem #5291) for 1 hour at 4° C. with constantstirring. Bacteria were then washed three times with 4° C. fresh PMBtreatment medium by centrifugation at 3,000×g for 10 minutes andresuspended at 2×10⁹ bacteria per mL. Bacteria recovery efficiency wasmonitored by following O.D.₆₀₀. Bacteria recovery after PMB treatmentand wash was greater than 90% for all samples treated with up to 300μg/mL PMB, and exceeded 80% for treatment with 1,000 μg/mL PMB.

In FIG. 1, endotoxin activity was determined by analyzing serialdilutions of untreated and treated bacterial cultures with the LimulusAmebocyte Lysate (LAL) Endosafe Endochrome-K kinetic assay kit (CharlesRiver Endosafe, Charleston, S.C.). Untreated cultures typicallycontained approximately 50-100 endotoxin units per 1×10⁶ bacteria.Similar endotoxin reductions were observed for treatment with 1,000μg/mL PMB in four independent experiments (average=17% of untreated).

In FIG. 2, bacterial viability was determined by serially diluting andplating each sample on LB Miller agar plates containing 2 mM MgCl₂, 0.5%glucose, with and without 1 mM DAP (to monitor viability andcontamination, respectively). Plates were incubated overnight at 37° C.,the number of colonies on each plate was determined, and then viabilitywas calculated by multiplying the number of colonies on each plate bythe dilution factor. The total number of bacteria in each suspension wascalculated by multiplying the O.D.₆₀₀ by the conversion factor of1.12×10⁹ bacteria/mL per O.D.₆₀₀ of 1. Viability (% Live Bacteria) wascalculated as the percent of viable bacteria/mL relative to the totalnumber of bacteria. Treatment with 1,000 μg/mL PMB reduced bacteriaviability to 0%. In subsequent scale-up experiments 1,000 μg PMB reducedviability to an average of 11% in four independent experiments.

Example 3

The experiments were carried out as described in Example 2, except thatpre-treatment washes, glutaraldehyde (GA) treatment and post-treatmentwashes were carried out with phosphate-buffered saline (PBS; Mg andCa-free) pH 7.5, containing 20 mM MgCl₂. Bacteria recovery after GAtreatment, at all concentrations tested, was typically 80-100%. FIG. 3demonstrates that treatment with 1% GA reduced endotoxin activity by96%. A 2-liter scale-up experiment with 1% GA treatment produced anendotoxin activity reduction of 82%, relative to the untreated culture.

Treatment with GA consistently produced 100% bacteria kill at dosesabove 0.05%, as demonstrated in FIG. 4.

Combination of 1,000 μg/mL PMB treatment followed by 1% GA treatmentusing 2 liters of late log phase culture produced bacteria with 0%viability and a 92% (12-fold) reduction in endotoxin activity, relativeto the untreated culture (Table 1).

Example 4

In Example 4, bacteria were grown and treated with 1,000 μg/mL PMB, 1%GA or both as described in the protocols for Examples 2 and 3. Sampleswere diluted with PBS, pH 7.5 containing 1% GA (if not previouslyexposed to GA) and fixed for 10 minutes. Twenty-five microliter dropletscontaining the bacteria were placed on parafilm and then covered with a100 mesh formvar+carbon EM grid (EMS, Hatfield, Pa.), which waspre-coated with 0.1% poly-L-lysine. Samples were allowed to adhere for10 minutes and then the grids were washed briefly three times byplacement on 200 microliter water droplets. The grids were negativelystained by placement for 1 minute on 100 microliter droplets of 2%uranyl acetate in water. Excess stain was blotted away with 3M filterpaper, followed by air drying. Samples were visualized using an FEITecnai Spirit G2 BioTWIN transmission electron microscope equipped witha bottom mount Eagle 4k (16 megapixel) digital camera (magnifications1,200× and 11,000×). The images in FIGS. 5B, 5C, and 5D confirm that PMBand/or GA treatments carried out according to the present methods leavethe bacteria intact, which is a desirable result. A polysaccharidecapsule is visible (fuzzy surface) on the untreated bacteria (FIG. 5A),but appears to have been removed or matted down in all treated bacteria(FIGS. 5B, 5C, and 5D).

Example 5

For Example 5, E. coli were grown and treated with 1,000 μg/mL PMB plus1% GA, and viability and endotoxin levels were determined as describedfor Examples 2 and 3. After final washing, untreated and PMB+GA-treatedbacteria were resuspended in 50% PBS, pH 7.5, 0.5 mM MgCl₂, 12%trehalose at a concentration of 1.1×10¹¹ bacteria per mL, aliquoted,flash frozen and stored at −80° C. The pyrogenicity threshold wasdetermined essentially as described in the United States Pharmacopeia,Chapter 151. Adult female New Zealand White rabbits weighing at least2.0 kg were used. All animals were conditioned with a sham test not morethan 7 days prior to the pyrogen test. Dose range-finding was carriedout with one rabbit per dose and these results were subsequentlyconfirmed with two rabbits per dose. Bacteria were diluted into sterilesaline for injection. All doses were delivered via the intravenous routein a volume of 10 mL. The lowest concentration of test agent thatproduced a temperature increase of 0.5-1.0° C. at any time point withinthree hours of test agent administration was considered to represent thepyrogenicity threshold. Rectal temperatures were recorded at baselineand at 30 minute intervals between 1 and 3 hours following injection oftest agent. Saline-diluted vehicle used for storage of untreated andtreated E. coli was shown not to be pyrogenic. Administration of 3×10⁴untreated bacteria to two rabbits produced temperature increases of 0.8and 1.0° C. Administration of 3×10⁵ PMB+GA-treated bacteria did notproduce a temperature increase of more than 0.1° C., but administrationof 9×10⁵ PMB+GA-treated bacteria to two rabbits produced temperatureincreases of 0.7 and 1.0° C., demonstrating a pyrogenicity thresholddifference of 30×-fold. It is likely that PMB neutralizes onlylipopolysaccharide-mediated pyrogenic activity. Whereas, GA mayneutralize pyrogenicity mediated by lipopolysaccharide. as well as byother constituents of the bacteria.

Table 1 demonstrates the pyrogenicity (febrile reaction) threshold foruntreated bacteria and bacteria treated with both 1,000 μg/mL PMB and 1%GA, as measured by a standard in vivo rabbit test. The results arecompared to endotoxin levels determined with the in vitro LAL assay,demonstrating that although PMB+GA treatment reduces endotoxin levels by12-fold, pyrogenicity mediated by the same sample is reduced by 30-fold,compared to untreated bacteria.

TABLE 1 Endotoxin Pyrogenicity Live Activity Threshold TreatmentBacteria LAL Assay Rabbit Assay No Treatment 83% 44.7 Units/10⁶ Bacteria3 × 10⁴ Bacteria PMB + GA  0%  3.6 Units/10⁶ Bacteria 9 × 10⁵ BacteriaMax Reduction 12X 30X

Example 6

In Example 6, E. coli were grown and treated with 1,000 μg/mL PolymyxinB plus 1% GA as described in the protocols for Examples 2 and 3. Frozenstocks of untreated and treated bacteria were thawed rapidly at 37° C.and either diluted at least 10-fold into sterile saline for injection(i.v. doses ≦3×10⁹ bacteria) or centrifuged at 3,000×g for 10 minutesand resuspended in sterile saline for injection (i.v. doses ≧5×10⁹).Bacteria or vehicles were injected i.v. via the tail vein in a volume of100 microliters.

Eight week old C57BL/6 or BALB/c female mice were used and acclimatedfor at least 7 days prior to studies. Mortality and clinicalobservations were performed once or twice per day. Additionalobservations were made at the time of and 1-4 hours after injections.Lack of toxicity by vehicles was confirmed. Cage side observationsincluded but were not limited to the following:

Changes in skin, fur, eyes, mucous membranes, gait, posture, andresponse to handling occurrence of secretions/excretions or otherevidence of autonomic activity such as lacrimation, piloerection,unusual respiratory patterns; presence of seizures; changes in generalalertness; stereotype behaviors such as excessive grooming andrepetitive circling; unusual behaviors (self-mutilating); development oflumps/bumps (tumor, abscess, etc.); development of signs of stressand/or respiratory symptoms; observation of the injection sites forsigns of irritation and inflammation; changes in food and waterconsumption and urine and feces output.

Bacteria administration for multiple-dose studies was carried out twiceper week for two weeks (4 treatments). Evaluation of toxicity includedmonitoring of animal weight. The mice used in the multiple-dose studyreported for PMB+GA-treated bacteria at 1×10⁹ were tumor-bearing. Allother mice reported in Table 2 were non-tumor bearing.

TABLE 2 Bacterial Single Dose Multiple (4) Dose Treatment DoseObservations Observations Untreated 3 × 10⁸ Slightly lethargic Slightlylethargic at 1-4 hr at 1-4 hr 1 × 10⁹ Lethargic at 1-4 hr Lethargic at1-4 hr, 2 of 3 mice dead after 4^(th) dose 5 × 10⁹ Lethargic at 1-48 hrND* Dead by 72 hr  1 × 10¹⁰ Dead by 18 hr ND PMB + 1 × 10⁹ Slightlylethargic Slightly lethargic at 1-4 hr, GA at 1-4 hr ruffled fur 3 × 10⁹Lethargic at 1-4 hr Slightly lethargic, lethargic or ruffled fur up to 4hr post treatment 5 × 10⁹ Lethargic at 1-4 hr Slightly lethargic,lethargic or ruffled fur up to 4 hr post treatment  1 × 10¹⁰ Severelylethargic, Lethargic, slightly lethar- 1 of 3 mice dead by gic, ruffledfur and/or 24 hr shallow breathing *ND = not determined

Example 7

In Example 7, 1,000 μg/mL PMB+1% GA-treated bacteria (DB103) wereprepared as described in the protocols for Examples 2 and 3. Eight weekold female C57BL/6J mice were shaved at the injection site and injectedsubcutaneously on the right flank with 2×10⁵ B16F10 murine melanomacells (ATCC CRL-6475). Treatments were started via tail vein i.v.administration three days later and continued twice per week for a totalof 5 treatments. DB103 in 50% PBS, pH 7.5, 0.5 mM MgCl², 12% trehaloseat a concentration of 1.1×10¹¹ per mL were diluted 11-fold (1×10⁹ dose)or 220-fold (5×10⁷ dose) with sterile saline and injected in a finalvolume of 100 microliters. The stock vehicle was diluted 11-fold for thevehicle control treatment group. Tumors were measured with caliperstwice weekly and tumor volume was determined using the formula(length×width²)/2. No compound-related deaths were observed. All animalsdeveloped tumors, with the exception of two animals treated with 1×10⁹DB103. Transient body weight loss of up to 3% (low dose group) and 7%(high dose group) was observed, but recovered after the last treatment(FIG. 6).

Example 8

For Example 8, E. coli (untreated and 1% GA-treated) were prepared asdescribed in the protocols for Examples 2 and 3. The experiment wascarried out as described in the protocol for Example 7, except thattreatment was started on day 11 when tumors were just palpable. Groupmeasurements were not recorded after day 24 for most groups because asubset of animals in each of these groups had to be euthanized due totumor burden. Tumors formed in all animals. Maximum weight loss in the1×10⁹ GA group was 11%. Toxicity precluded administration of 1×10⁹untreated E. coli (see Table 2).

Example 9

In Example 9, 1,000 μg PMB+1% GA-treated bacteria (DB103) were preparedas described in the protocols for Examples 2 and 3. The experiment wascarried out as described in the protocol for Example 7, except that1×10⁵ murine CT26 colorectal carcinoma cells were injectedsubcutaneously in the right flank of BALB/c mice. DB103 treatments werestarted via tail vein i.v. administration three days later and continuedtwice per week for a total of 6 treatments. Cyclophosphamide (LKTLaboratories, #C9606) was administered via the drinking watercontinuously, starting on day 3, at ˜20 mg/kg/day (0.133 mg/mL inwater). Anti-murine CTLA-4 antibody (BioXcell #BE0164), 100 μg in 200microliters PBS, was administered i.p. on days 3, 6 and 9. Clinicalobservations and mortality were recorded daily. Tumors were measuredwith calipers twice weekly and tumor volume was determined with theformula (length×width²)/2. Tumors formed in all mice in the vehiclegroup. No weight loss and no compound-related deaths were observed inany group. The data for the vehicle, low dose and high dose DB103 groupsis the same in FIGS. 8A and 8B.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe disclosure pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The disclosure illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the claims. Thus, it should be understood that although thepresent disclosure has been specifically described by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.Other embodiments are set forth within the following claims.

What is claimed:
 1. A method for treating a cancer comprisingadministering to a mammal with cancer a composition that comprises anamount of intact and substantially non-viable Gram-negative bacterialcells, wherein the bacterial cells are treated with polymyxin B underconditions to reduce viability, endotoxin activity and pyrogenicity ofthe cells without loss of cell integrity such that the cells have asubstantial reduction in endotoxin activity and pyrogenicity as comparedto untreated bacterial cells, and wherein the amount administered issufficient to decrease growth or metastasis of the cancer.
 2. The methodof claim 1, wherein the bacterial cells are treated with radiation. 3.The method of claim 2, wherein the mammal is a human.
 4. The method ofclaim 1, wherein the cancer is a solid tumor.
 5. The method of claim 1,wherein the mammal is further administered an antagonist of an immunefunction inhibiting T-cell receptor or T-cell receptor ligand selectedfrom the group consisting of CTLA-4, PD-1, PD-L1 and PD-L2.
 6. Themethod of claim 1, wherein the mammal is further administered an agonistof an immune function stimulating T-cell receptor selected from thegroup consisting of GITR, 4-1BB, CD40 and OX40.
 7. The method of claim1, wherein the mammal is further administered a chemotherapeutic agent.8. The method of claim 7, wherein the chemotherapeutic agent iscyclophosphamide.
 9. The method of claim 1, wherein the mammal isfurther administered a cytokine.
 10. The method of claim 1, wherein theGram-negative bacterial cells are Salmonella cells.
 11. The method ofclaim 1, wherein the Gram-negative bacterial cells are Escherichiacells.
 12. A method for treating a cancer comprising administering to amammal with cancer a composition that comprises an amount of intact andsubstantially non-viable Gram-negative bacterial cells, wherein thebacterial cells comprise a (i) genetic defect that disrupts thebiosynthesis of KDO2-Lipid IV_(A) or (ii) a genetic mutation thatprevents O-acylation of KDO2-Lipid IV_(A) sufficient to substantiallyreduce endotoxin activity and pyrogenicity and wherein the amountadministered is sufficient to decrease growth or metastasis of thecancer.
 13. The method of claim 12, wherein the defect is in the msbB orlpxM loci.
 14. A method for treating a cancer, comprising: treatingGram-negative bacterial cells with polymyxin B under conditions toreduce viability, endotoxin activity and pyrogenicity of the cellswithout loss of cell integrity, to obtain a plurality of intact andsubstantially non-viable Gram-negative bacterial cells with substantialreduction in endotoxin activity and pyrogenicity as compared tountreated bacterial cells, and administering the plurality of intact andsubstantially non-viable Gram-negative bacterial cells to a cancerpatient.